Single-Cell Transcriptome Analysis of Olfactory Sensory Neurons

Olfactory sensory neurons (OSNs), which detect a myriad of odorants, are known to express one allele of one olfactory receptor (OR) gene (Olfr) from the largest gene family in the mammalian genome. The OSNs expressing the same OR project their axons to the main olfactory bulb where they converge to form glomeruli. This “One neuron-one receptor rule” makes the olfactory epithelium (OE), which consists of a vast number of OSNs expressing unique ORs, one of the most heterogeneous cell populations. However, the mechanism of how the single OR allele is chosen remains unclear along with the question of whether one OSN only expresses a single OR gene, a hypothesis that has not been rigorously verified while we performed the experiments. Moreover, failure of axonal targeting to single glomerulus was observed in MeCP2 deficient OSNs where delayed development was proposed as an explanation for the phenotype. How Mecp2 mutation caused this aberrant targeting is not entirely understood.

In this dissertation, we explored the transcriptomes of single and mature OSNs by single-cell RNA-Seq to reveal their heterogeneity and further studied the OR gene expression from these isolated OSNs. The singularity of sequenced OSNs was ensured by the observation of monoallelic expression of X-linked genes from the hybrid samples from crosses between mice of different strains where strain-specific polymorphisms could be used to track the allelic origins of SNP-containing reads. The clustering of expression profiles from triplicates that originated from the same cell assured that the transcriptomic identities of OSNs were maintained through the experimental process. The average gene expression profiles of sequenced OSNs correlated well to the conventional transcriptome data of FACS-sorted Omp-positive cells, and the top-ranked expression of OR was conceded in the single-OSN transcriptomes. While exploring cellular diversity, in addition to OR genes, we revealed nearly 200 differentially expressed genes among the sequenced OSNs in this study. Among the 36 sequenced OSNs, eight cells (22.2%) showed multiple OR gene expression and the presences of additional ORs were not restricted to the neighbor loci that shared the transcriptional effect of the primary OR expression, suggesting that the “One neuron-one receptor rule” might not be strictly true at the transcription level. All of the inferable ORs, including additional co-expressed ORs, were shown to be monoallelic. Our sequencing of 21 Mecp2308 mutant OSNs, of which 62% expressed more than one OR genes, and the expression levels of the additional ORs were significantly higher than those in the wild-type, suggested that MeCP2 plays a role in the regulation of singular OR gene expression. Dual label in situ hybridization along with the sequence data revealed that dorsal and ventral ORs were co-expressed in the same Mecp2 mutant OSN, further implying that MeCP2 might be involved in regulation of OR territories in the OE. Our results suggested a new role of MeCP2 in OR gene choice and ratified that this multiple-OR expression caused by Mecp2 mutation did not accompany delayed OSN development that has been observed in the previous studies on the Mecp2 mutants.

Association between the oxytocin receptor (OXTR) gene and mesolimbic responses to rewards.

BACKGROUND: There has been significant progress in identifying genes that confer risk for autism spectrum disorders (ASDs). However, the heterogeneity of symptom presentation in ASDs impedes the detection of ASD risk genes. One approach to understanding genetic influences on ASD symptom expression is to evaluate relations between variants of ASD candidate genes and neural endophenotypes in unaffected samples. Allelic variations in the oxytocin receptor (OXTR) gene confer small but significant risk for ASDs for which the underlying mechanisms may involve associations between variability in oxytocin signaling pathways and neural response to rewards. The purpose of this preliminary study was to investigate the influence of allelic variability in the OXTR gene on neural responses to monetary rewards in healthy adults using functional magnetic resonance imaging (fMRI). METHODS: The moderating effects of three single nucleotide polymorphisms (SNPs) (rs1042778, rs2268493 and rs237887) of the OXTR gene on mesolimbic responses to rewards were evaluated using a monetary incentive delay fMRI task. RESULTS: T homozygotes of the rs2268493 SNP demonstrated relatively decreased activation in mesolimbic reward circuitry (including the nucleus accumbens, amygdala, insula, thalamus and prefrontal cortical regions) during the anticipation of rewards but not during the outcome phase of the task. Allelic variation of the rs1042778 and rs237887 SNPs did not moderate mesolimbic activation during either reward anticipation or outcomes. CONCLUSIONS: This preliminary study suggests that the OXTR SNP rs2268493, which has been previously identified as an ASD risk gene, moderates mesolimbic responses during reward anticipation. Given previous findings of decreased mesolimbic activation during reward anticipation in ASD, the present results suggest that OXTR may confer ASD risk via influences on the neural systems that support reward anticipation.